The proposed research is in two distinct areas, sequence dependent nucleic acid dynamics as studied using EPR spectroscopy and nucleic acid interactions with bifunctional alkylating agents. In the former area, we will continue our efforts to synthesize useful nitroxide spin probes for EPR studies of nucleic acid dynamics. These studies provide information on the accessibility of non-ground state conformations of DNA which may be important in its recognition by proteins and small molecules. The target spin probe, which it is proposed will overcome a substantial deficiency in currently available spin probes, will be tested for structural compatibility with B-DNA, will be compared using EPR techniques to a probe developed during the previous grant period, and will be used to prepare a spin labeled hairpin, four-arm junction (Holliday intermediate), mismatches, and interstrand cross-linked DNA for use in dynamics studies. The proposed studies in the area of bifunctional DNA alkylating agents will focus on the study of DNA interstrand cross-linking reactions of the antitumor substances FR900482 and FR66979, 2,5-bis-(N-aziridinyl)-1,4-benzoquinone (DZQ), and the bioregulatory substance nitric oxide. Interstrand cross-linking is commonly cited as a toxic mechanism for several agents presently used in cancer chemotherapy. An understanding of their agents, we will define the consensus sequence for cross-linking, the functional groups required for cross-linking, and the covalent structures of the relevant lesions. We will use the Crothers PAGE retardation method to assess bending and torsional reorganization in a wide variety of interstrand cross-linked DNAs. In collaboration with Professor Reid, we will seek to determine the three dimensional solution structure of an interstrand cross-linked DNA.